Project summary: There is a fundamental gap in our understanding of the mechanism by which the highly pathogenic Ebola and Marburg filoviruses enter their host cells and initiate infection. The long-term goal is to better understand, in molecular detail, the interactions between virus and host that are necessary for filoviruses to invade cells and organisms. The objective of this particular application is to understand how filoviruses exploit endosomal cysteine cathepsins, a class of cysteine proteases recently identified by us to be essential host factors, to enter cells. The central hypothesis is that the viral glycoprotein, GP undergoes a multistep program of proteolytic disassembly mediated by cysteine cathepsins, that is required for, and culminates in, triggering of the membrane fusion activity of GP. The membrane fusion reaction thus initiated, results in coalescence of viral and cellular membranes, and delivery of the viral nucleocapsid into the host cell cytoplasm. Guided by strong preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) Identifying the cysteine cathepsins required for filovirus entry; 2) Identifying the sequence and structural determinants of GP proteolytic disassembly by cysteine cathepsins;and 3) Determining the role of cysteine cathepsin-mediated GP disassembly in entry Relevance to public health: Ebola and Marburg viruses are the cause of an invariably fatal hemorrhagic fever in humans in central Africa, and are also potential agents of bioterrorism. This work has potential applicability to developing antiviral countermeasures for these highly pathogenic agents.